SUMMARY/ABSTRACT This proposal focuses on the development of a high-throughput, high sensitivity, low blood-volume, multiplex assay for in vitro diagnosis of allergies termed AllerBeadTM. Allergies comprise a group of immune disorders constituting the 6th leading cause of chronic disease in the US. In the case of pediatric allergies, over 50 million children are affected, a number which is rapidly increasing, making it the most common chronic childhood disease. Early identification of allergen triggers can be life-saving and a key to managing the debilitating symptoms which can limit a child's activities, as well as critical for developing treatments including new forms personalized desensitizing therapies. Utilization of in vitro allergen-specific IgE (sIgE) testing, especially in pediatrics, has many advantages over the more common in vivo skin prick test (SPT) including: i) lower costs, ii) elimination of possible adverse reactions and patient intolerance of the tests, particularly in pediatric subjects, iii) highly standardized assay procedures and allergen preparations used in the tests and iv) objective/quantitative readout of sIgE levels as well as automation and high throughput capability. In recognition of the significant market for pediatric in vitro diagnostic allergy testing, several companies have sought to develop multiplex systems designed to use only a few drops of blood to assay multiple allergens and allergen component proteins. For example, one approach based on use of protein microarray technology [2, 3] is capable of measuring ~100 allergen components simultaneously. However, limitations of protein microarrays including in sensitivity, reproducibility, scalability, non-linearity and expense of manufacture prevents this approach from replacing the more widely used one-by-one allergen assays such as ELISA and Phadia's ImmunoCAP(R) in the pediatric market. In contrast, AmberGen has developed and evaluated during Phase I a high sensitivity, high-throughput, low cost multiplex immunoassay approach by adapting the VeraCode(R) digital holographic micro-bead technology developed by Illumina for genomic applications. The heart of this new approach relies on proprietary methods developed by AmberGen including the use of cell-free protein expression for uniform and reproducible production of highly pure allergen components, low immunogenic 1-step protein attachment to the bead surface and elimination of interfering IgGs using photocleavable antibodies. Significantly, Phase I results based on a multiplex assay of 27-allergens from the 8 major food groups achieved all 3 major aims. This work demonstrated that AllerBeadTM results in sensitivities and CVs similar to the gold-standard FDA-approved ImmunoCAP(R) assays for individual allergens, but is capable of automatically and rapidly assaying 96- samples for up to 384 individual allergens from only 10 L of blood per patent. During Phase II, the most comprehensive and low cost blood test for childhood food allergies comprising 60- allergen proteins and whole food extracts, including all of the Big 8 food groups which account for >90% of all childhood food allergies, will be developed. Results using AllerBeadTM technology will be compared with measurements obtained commercially from the non-multiplexed ImmunoCAP(R) assay and also on the multiplexed Phadia ISAC(R) Microarray. For the purpose of assay validation and eventual FDA approval, AmberGen will work closely with INOVA Diagnostics and collaborate with Dr. Lynda Schneider, M.D., Director of the Allergy Program at Children's Hospital, Boston, Associate Professor of Pediatrics, Harvard Medical School, who will provide 900 validated blood samples under IRB approval. Dr. Paul Billings, the Chief Medical Officer at Life Technologies and Dr. Kevin Xie, former Head, Global Strategic Business Development at Boehringer-Ingelheim and at Bayer Healthcare will also serve as consultants.